The Alkaline Phosphatases (ALPs) are a group of functionally similar enzymes which are found in nearly all organisms. They are magnesium and zinc containing enzymes and are inhibited by metal chelators. ALPs function to hydrolyze monophosphate esters such as phosphate esters of primary alcohols and phenols. Phosphates inhibit ALP activity. The hydrolysis reaction has an alkaline optimum pH. Whenever the term ALP or ALPs are used it can mean any one of these families of enzymes.
In organisms exhibiting tissue differentiation, ALPs appear in many of the different tissues and are named after the tissue of origin. ALPs originating from different tissues show slight differences in stability, catalytic properties and susceptibility to inhibition to a variety of inhibitors. Fishman, W. H. Alkaline Phosphatase Isozymes: Recent Progress, Clinical Biochemistry 1990; 23: pp. 99-104. For example, human ALPs from bone, liver and kidney show differential mobility on electrophoresis due to differences in their sialic acid content. Moss, D. W., Alkaline Phosphatase Isoenzymes, Clinical Chemistry 1982; 28: pp. 2007-2016.
Because of their enzymatic nature, ALPs have been used as a component in solid phase diagnostic assays as the label or indicator molecule to detect the presence or concentration of an analyte contained in a human bodily fluid sample. The label can be covalently attached to an analyte (or an analogue of the analyte) and the labeled analyte competes for a limited number of analyte receptors (e.g. antibodies) in a competitive assay format. Alternatively a label can be covalently attached to a second analyte receptor in a sandwich assay format. In both cases the label with its covalently attached analyte or analyte receptor is often called a conjugate.
The label of a conjugate must directly or indirectly produce a measurable signal. For example, the label of the conjugate can be a fluorescent or calorimetric compound or it can be an enzyme which produces a fluorescent, electrochemical, chemiluminescent, thermometric, or colorimetric signal when the enzyme reacts with a substrate molecule. The amount of signal formed is correlated with the amount of analyte in a test sample.
Enzymes are used as labels in immunoassay systems because of their amplifying effect. A single molecule of enzyme typically converts 10.sup.3 to 10.sup.4 molecules of substrate into product per minute. The product of an enzyme-substrate reaction can be measured calorimetrically, fluorometrically or by any other quantifiable methods. Ideally, enzymes should have a high catalytic activity at low substrate concentration; the enzyme should be stable at the pH required for receptor-analyte binding; the enzyme should have reactive groups through which the enzymes can be covalently linked with a minimum loss of activity; the enzyme should be stable under routine storage and assay conditions; and a test sample should not have enzyme activity.
Calf intestinal ALP is the ALP most commonly used as a label. It has a large number of free amino groups which can be used for conjugation without loss of enzyme activity. It has good stability in commonly used buffer systems at ambient temperatures and also possesses high temperature stability. Its optimum activity is seen in the pH range 9.5-10.5 but maximum signal is a complex function of buffer composition, ionic strength, pH, substrate and substrate concentrations. The buffer system for the ALP-substrate reaction is often diethanolamine or Tris and usually includes a magnesium salt such as magnesium chloride and/or zinc salts such as zinc acetate. Common substrates are para-nitrophenyl phosphate (p-NPP), 4-methylumbelliferyl phosphate (4-MUP) and 3-(2'-spiroadamantane)-4-methoxy-4-(3"-phosphoryloxy) phenyl-1,2-dioxetane (AMPPD). Some active ALP is present in normal human bodily fluids and may contribute to background signal. Generally specific binding is seen in various assay formats but is particularly evident in solid phase assays. See, Gorman, Eileen et al. An overview of automation, Principles and Practice of Immunoassay, Price, C. and Newman, D., Editors, Stockton Press; 1991: p. 234-236.
Methods have been developed to reduce non-specific binding. In solid phase assay formats one method has been to wash the reaction zone (i.e. area of the solid phase support having the receptor-analyte complex thereon) of the solid phase prior to addition of the substrate molecule with a wash solution containing detergents such as Tween or Brij. The solution may also contain salts, proteins such as Bovine Serum Albumin (BSA), or denaturants.
Another approach to reduce the effects of the human ALPs is to use inhibitors of ALPs in the substrate solution. This approach is especially useful in cases where the reaction zone of the solid phase support is not washed to remove the non-specifically bound interfering substances. For example, use of an inhibitor with unique K.sub.i properties could potentially reduce the effect of the human ALPs. K.sub.i is the concentration of the inhibitor which decreases enzyme activity by 50% (i.e. the lower the K.sub.i, the more effective the inhibitor). Ideally the inhibitor should have a high K.sub.i for the ALP used in the conjugate, but a low K.sub.i for human ALPs that may be present in disease states. Common inhibitors to ALPs include L-phenylalanine, homoarginine, tetramisole and leva- misole and derivatives thereof. Recently 5,6-Dihydro-6-(2-naphthyl)imidazo-2,1-b!thiazole has been used as an inhibitor for human ALPs. The last compound, with a K.sub.i of about 1.0 uM has been shown to be most effective as an inhibitor of ALPs in Sarcoma 180/TG ascites cells. See for example, Bhargava K. K. et al, Tetramisole analogues as inhibitors of Alkaline Phosphatase. an enzyme involved in the resistance of Neoplastic cells to 6-Thiopurines, J. Med. Chem. 1977; 20: pp. 563-566. However, even this inhibitor alone may not completely eliminate the effects of the human ALPs of different origins.
Some human bodily fluid test samples have been found to contain elevated levels of the various forms of normal molecular weight ALPs. These elevated levels are associated with a number of clinical symptoms and disease states. See for example, Narayanan S., Serum Alkaline Phosphatase Isoenzymes as Markers of Liver Disease, Annals of Clinical and Laboratory Science 1991; 21: pp. 12-18; Severim G. et al, Diagnostic aspects of alkaline phosphatase: separation of isoenzymes in normal and pathological human serum by high-performance liguid chromatography, Journal of Chromatography 1991; 563: pp. 147-152 and Harmenberg U. et al, Identification and Characterization of Alkaline Phosphatase Isozymes in Human Colorectal Adenocarcinomas, Tumor Biology 1991; 12: pp. 237-248.
Higher molecular weight forms of ALPs have been found in some human bodily fluid test samples and also have been found to be associated with many of these disease states. See for example, Wei J. S. et al, Quantitative determination of high molecular weight alkaline Phosphatase in patients with colorectal cancer by polyacrylamide gel electrophoresis, Enzyme 1990; 43: pp. 188-191 and Kihn L. et al, High-Molecular-Weight Alkaline Phosphatase in Serum Has ProDerties Similar to the Enzyme in Plasma Membranes of the Liver, Clinical Chemistry 1991; 96: pp. 470-478. The nature and origin of high molecular weight forms of ALP in disease states is not clear. Questions have been raised on whether it is a single species or whether it is ALP of normal molecular weight complexed with membrane particles or antibodies. In any event, the higher molecular weight forms are difficult to remove from the solid phase.
In diagnostic assays, human bodily fluid test samples that contain the higher molecular weight ALP or elevated levels of normal molecular weight ALP result in a related, although distinct and more serious form of non-specific binding. This problem will be referred to as serum-mediated non-specific binding. See, Gorman, Eileen et al. An overview of automation, Principles and Practice of Immunoassay, Price, C. and Newman, D., Editors, Stockton Press; 1991: p. 234. The ALP of these samples can contribute not only to the background signal of the diagnostic assay, but also can produce a falsely positive result even in the absence of the analyte of interest.
Current wash compositions utilized in solid phase diagnostic assays may have included detergents and inhibitors but are not completely effective in reducing serum-mediated binding. In particular, high molecular weight ALPs have poor washing qualities and are either not removed or only partially removed from the reaction zone of the solid support.
If the effects of the human ALP are not reduced, the human ALP of these test samples can react with the substrate molecule to produce additional signal. This will contribute to the signal generated from the ALP of the conjugate. This excess signal will lead to erroneous results in the determination of the analyte concentration. These false positive results are difficult to detect, except by comparison to a non-ALP reference assay or by factoring in the patient's medical history.
Thus, a wash solution is needed that will be stable, reliable, and effective in removing or reducing the effect of human high molecular weight ALP and elevated levels of human ALP from the reaction zone of a solid phase assay.
A continuing need exists for improved methods and compositions to correct the above deficiencies.